The surgical specimens from 41 patients who had primary surgical treatment of HCC at Guilin Medical University Affiliated Hospital between 2009 and 2010 were studied. These surgical specimens included the primary HCC lesion and its corresponding adjacent tissue (2 cm away from the edge of the tumor lesion). All cases were pathologically confirmed, and the patients had no prior treatment before surgery. Forty cases of normal adjacent liver tissues from liver trauma and hepatic hemangioma were used as the control tissues. All of the samples were collected with approval from the Medical Ethics Committee at Guilin Medical University Affiliated Hospital.

LO2, HepG2, and Hep3B cells (American Type Culture Collection, USA) were cultured in RPMI-1640 medium (Gibco-BRL, NY, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc., UT, USA) and 100 U/ml of penicillin plus 100 U/ml of streptomycin in a 37°C incubator with 5% CO₂ and 95% air. MHCC97-H and MHCC97-L cells (American Type Culture Collection) were cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (Hyclone Laboratories) and 100 U/ml of penicillin plus 100 U/ml of streptomycin in a 37°C incubator with 5% CO₂.

The paraffin sections were dewaxed with xylene and rehydrated in descending concentrations of ethanol. The endogenous peroxidase was inhibited, and the slides were incubated with antibodies against DOR (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated at 4°C overnight in a humidified container. After washing with PBS three times, the tissue slides were treated with a non-biotin horseradish peroxidase detection system according to the manufacturer’s instructions (Dako).

Cells in the logarithmic growth phase were digested with 0.25% trypsin. The cells were seeded on coverslips inside the wells at a concentration of 1×10⁵ cells/ml and incubated for 24 h. After the incubation, the old medium was discarded, and the cells were washed three times in PBS. Paraformaldehyde (4%) was used to fix the cells for 30 min, followed by three washes in PBS to remove the excess fixative. Cells were then incubated with 0.5% Triton X-100 for 20 min at room temperature (RT), washed three times with PBS, and blocked using 5% goat serum at RT for 30 min. After removing the serum, a rabbit anti-human DOR monoclonal antibody (1:500 dilution) was added and incubated at 37°C for 2 h. The cells were washed three times in PBS for 5 min each before incubating with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit secondary antibody (1:200 dilution) at 37°C for 30 min. After three washes in PBS for 5 min each, all of the coverslips were collected and mounted onto glass slides using glycerol. All of the samples were analyzed using fluorescent microscopy. The primary antibody was omitted in the negative control.

Cells during the logarithmic growth stage were digested using 0.25% trypsin. Cells were seeded into a 96-well plate at a concentration of 1×10⁴ cells/ml and incubated at RT for 24 h. After the cells attached, different doses of naltrindole (Sigma, USA) were added to 6-wells for each dose. The negative control group had no naltrindole treatment. The plate was incubated in a 5% CO₂ incubator for 48 h and then 20 μl of MTT (5 mg/ml) was added and incubated in a CO₂ incubator for 4 h at 37°C. After the incubation, the excess liquid was discarded and the cells were incubated with 150 μl of DMSO at RT for 10 min on a shaker. The OD₅₇₀ value was measured using a microplate reader.

Cells were digested in trypsin and centrifuged to collect the cell pellets. The cell pellets were washed three times in PBS, fixed in pre-chilled 70% ethanol and then stored at 4°C overnight. The next day, the cells were washed three times with PBS and resuspend into 100 μl of PBS at a final concentration of 1×10⁶ cells/ml. Comprehensive DNA Stain (500 μl) (RNase 50 mg/l, propidium iodide (PI) 100 mg/l and Triton X-100 1 ml/l) was added to the cell solution followed by light protected incubation at RT for 15 min. The labeled cells were analyzed using flow cytometry.

During the early stage of apoptosis, the loss of cell membrane symmetry causes phosphatidylserine (PS) to be exposed on the outside of the cell membrane. Annexin V-FITC can bind specifically with PS on the intact cell membrane. Therefore, Annexin V-FITC staining can be used to detect early stage apoptotic cells in a faster and more sensitive way. The cells were digested in 0.25% trypsin and then incubated at a concentration of 1×10⁶ cells/ml with 5 μl of Annexin V-FITC and PI. The cells were further incubated protected from light at 37°C for 15 min and analyzed using flow cytometry.

The cells were cultured for 24 h in a 6-well plate with poly-L-lysine pre-coated coverslips. After apoptosis induction, the medium from the cell culture was removed. The cells were fixed in 4% paraformaldehyde for 30 min. The fixing solution was then removed and the cells were washed with PBS twice for 3 min each. After washing, 5 μg/ml of Hoechst 33342 dye was added to each sample. The plate was incubated at 37°C for 10 min. The staining solution was removed followed by two washes in PBS for 3 min each. One drop of fluorescence quencher was added to the slide. The coverslip with the cells attached was exposed to the fluorescence quencher and mounted onto the slide carefully to avoid bubbles. The slides were observed and documented using fluorescent microscopy.

Total RNA was extracted as previously described (22) and the total RNA concentration was measured. The primer sequences for DOR and β-actin are listed in Table I. RT-PCR was performed according to the RT-PCR kit manual (Takara Bio Inc.). The PCR products were analyzed using 1.0% agarose gel electrophoresis and a gel imaging system.

Cells at a concentration of 5×10⁴ cells/ml were seeded into a 6-well plate. After reaching 70% confluence, the cells were transfected with lentiviral vectors according to the transfection agent manual. The short interfering RNA (siRNA) sequences targeting DOR (synthesized by Invitrogen) are listed in Table II. The RNA interference efficiency was tested using RT-PCR and western blotting.

Cells were harvested and lysed in 2 ml of lysis buffer [50 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% NP-40 and 5 mM cocktail solution (pH 7.4)] to extract the proteins. The extracted proteins were quantified using the BCA method and analyzed using SDS-PAGE. The extracted proteins were transferred onto a PVDF membrane by semi-dry transfer. The membrane was blocked in 5% non-fat milk overnight at 4°C. The membrane was washed using TBST and incubated with primary antibody at 37°C for 1 h, washed with TBST and then incubated with secondary antibody at 37°C for 1 h. The membrane was washed with TBST and developed for 5 min using autoradiography. Quantity One software was used to analyze the optical density. The results are displayed as the ratio of the optical density value/internal reference optical density value.

Cells were incubated in a 6-well plate to 100% confluence. A sterilized tip was then used to scratch a line in the center of the monolayer cells. The dead cells were removed by washing and cell growth was observed and documented at different time-points (0, 24, 48 and 72 h) under a microscope. Image-pro Plus software was used to measure the distance between the scratch edges at different time-points (0, 24, 48 and 72 h). The cell migration distance at different time-points was measured based on the following equation: Distance at 0 h / Distance at 24, 48, or 72 h. The results were analyzed using statistical software.

Transwell migration chambers (Corning, NY, USA) were used for the cell invasion test. Different groups of MHCC97-H cells were digested using trypsin. Cell counting was performed and the cells were then resuspended to a final concentration of l×10⁵ cells/ml in DMEM media. Resuspended cells (200 μl) were added to the upper chamber in each Transwell (8-μm diameter pore size) and 500 μl of medium supplemented with serum was added to the lower chamber. Transwell migration chambers were incubated in a 5% CO₂ incubator for 24 h, washed three times with PBS and then stained with crystal violet. Five fields were randomly picked and the cell numbers were counted in every field using a light microscope at ×200 magnification. The mean value of the cell numbers in the five fields was used as the number of cells that passed through the artificial human basal membrane.

The animal experiments were approved by the Medical Ethics Committee at Guilin Medical University. Ten nude mice (male, 6–8-week-old and weighing ∼20 g) were purchased from the Animal Center at Guilin Medical University. The mice were randomly divided into two groups with five mice in each group. All of the mice were spleen inoculated with cells (0.5l×10⁵ cells per mouse) transfected with control oligonucleotides (N-control) or DOR-siRNAs. Animals were sacrificed after four weeks using cervical dislocation and the xenograft tumor was harvested aseptically for testing.

SPSS 16.0 statistical software was used to perform all of the statistical analyses. The data are the mean ± standard deviation. Both a one-way ANOVA and an LSD-t test were used to compare the differences among the different groups. The difference between two groups was considered to be statistically significant at p<0.05.

The RT-PCR results demonstrated variable levels of DOR expression in 41 cases of HCC samples. The DOR mRNA levels in HCC lesions were higher than in the adjacent tumor tissues and normal liver tissues (p<0.05) (Fig. 1A). The DOR mRNA was expressed in different types of HCC cells at levels that were significantly higher than in normal liver cells (p<0.05) (Fig. 1B). Western blot analysis determined that DOR protein expression in HCC tissue was higher than in the adjacent tumor tissues and normal liver tissues (p<0.05) (Fig. 1C). Moreover, DOR protein expression in different types of HCC cells was higher than in normal liver cells (p<0.05) (Fig. 1D).

Immunohistochemical detection of DOR expression and distribution of human hepatocellular carcinoma were carried out. The results show that the DOR positive staining in HCC are mainly located in the cell membrane, a small amount of staining in the cytoplasm and none in the nucleus (Fig. 1E). Immunofluorescence using FITC-conjugated antibodies was used to detect DOR expression and its distribution in HCC and normal cells. The results from the HCC cells showed that the green fluorescence FITC signal was distributed evenly on the cancer cell membrane, was weak in the cytoplasm and was absent in the nucleus (Fig. 1F), indicating that DOR was mainly expressed on the HCC cell membrane.

HepG2 and Hep3B HCC cell lines were employed to study the effect of DOR on HCC cell proliferation. An MTT assay was used to quantify the cell proliferation rate after DADLE treatment. When the DADLE concentration was increased from 10 nM to 10 μM, the OD₅₇₀ values of the DADLE-treated HepG2 and Hep3B cells also increased, but there was no increase in the control groups (without DADLE treatment). However, when the concentration of DADLE was above 1.0 μM, the OD₅₇₀ values of HepG2 and Hep3B cells did not increase further, indicating that the activation of DOR can promote HCC cell proliferation and that 1.0 μM of DADLE induces maximal cell proliferation (Fig. 2).

Three siRNAs (DOR-siRNA-1, DOR-siRNA-2 and DOR-siRNA-3) and the control siRNA (N-control) were used to transfect HCC cells. Post-transfection, the total RNA was extracted for use in the RT-PCR analysis of DOR expression in HCC cells. Upon siRNA transfection, DOR expression decreased relative to cells transfected with the control siRNA. DOR-siRNA-1 gave the most obvious silencing effect (Fig. 3A). Western blot analysis further illustrated that DOR protein expression also decreased after DOR-siRNA-1 transfection (Fig. 3B).

OD₅₇₀ values of DOR-siRNA-transfected HepG2 and Hep3B cells were significantly reduced relative to those from the HepG2 and Hep3B cells transfected with the control siRNA (p<0.05). Similar results were observed in naltrindole (specific DOR inhibitor) treated cells (p<0.05) (Fig. 4), suggesting that the downregulation of DOR can significantly suppress HCC cell proliferation.

To study the function of DOR in HCC cellular apoptosis, Hoechst 33342 staining was used to measure the effect of the downregulation of DOR on apoptotic HCC cell morphology using fluorescence microscopy. The HCC cell nucleus in the N-control group was circular or oval in shape with the chromatin evenly distributed in light blue fluorescence. The silencing of the DOR gene or the inhibition of DOR using the specific antagonist naltrindole led to a significant increase in the number of apoptotic cells. The chromatin in the apoptotic HCC cells was condensed and unevenly distributed. Chromatin accumulation near the nuclear membrane, chromatin condensation, increased intensity of fluorescence staining, nuclear condensation and apoptotic cell bodies were also detected when DOR was downregulated (Fig. 5A).

The Annexin V-FITC/PI double-labeling method was used to study the effect of DOR downregulation on the HCC cell apoptotic rate. DOR silencing increased the rate of early apoptosis in HepG2 and Hep3B cells relative to the N-control group (p<0.05). We also observed that the rate of early apoptosis in the HepG2 and Hep3B cells increased with naltrindole treatment (p<0.05) (Fig. 5B and C). These results suggest that the downregulation of DOR can promote apoptosis in HCC cells.

Flow cytometry analysis was used to determine whether DOR affected the cell cycle in HCC cells. When the DOR gene was silenced using siRNAs or the cells were treated with naltrindole, the cell cycle of HepG2 and Hep3B cells was arrested at G0/G1 (p<0.05), suggesting that the downregulation of DOR increased the percentage of HCC cells in G0/G1. This down-regulation also inhibited HCC cell growth (Fig. 6).

To determine if the downregulation of DOR can affect HCC cell invasion and migration, we compared the number of cells passing through an artificial human basal membrane before and after siRNA transfection. Five fields of cells were randomly chosen for this analysis. The downregulation of DOR decreased the invasion ability of HCC cells and significantly reduced the number of cells passing through the artificial human basal membrane (p<0.05). Moreover, the downregulation of DOR also reduced the migration of HCC cells (p<0.05) (Fig. 7A and B).

The cell monolayer was scratched and the distance between scratch margins was measured at 0, 24, 48 and 72 h. The downregulation of DOR led to a significant decrease in the migration of HCC cells at the four different time-points (p<0.05), which suggested that the downregulation of DOR led to a significant reduction in liver cancer cell migration (Fig. 7C and D).

After four weeks of inoculation, the tumor formation rates reached 100% in all nude mice (n=5) in the N-control group and 80% in the nude mice inoculated with the DOR-siRNA-transfected cancer cells. Tumor progression in the nude mice of the N-control group was more rapid than in the nude mice inoculated with DOR-siRNA-transfected cancer cells (Fig. 8A). We also found that the tumor volumes in the DOR-siRNA group were significantly smaller than in the N-control group (p<0.05) at all time-points (Fig. 8B). The tumor weights of the DOR-siRNA group were significantly smaller than the weights of the N-control group (Fig. 8C). These results suggested that the downregulation of DOR in vivo can significantly decrease cancer progression.